Flow cytometric detection and enumeration of DNA and RNA viruses infecting marine eukaryotic microalgae

Sample preparation protocols for flow cytometry (FCM) analysis with five algal viruses were optimized: Heterocapsa circularisquama virus (HcV), Heterosigma akashiwo virus (HaV), Chaetoceros salsugineum nuclear inclusion virus (CsNIV), Rhizosolenia setigera RNA virus (RsRNAV) and H. circularisquama RNA virus (HcRNAV). The optimum staining protocols differed significantly among the viruses tested. FCM counts for the large DNA algal viruses HaV and HcV (∼0.2 µm in diameter) were similar to numbers determined by epifluorescence microscopy (EFM). In contrast, FCM counts of viruses smaller than 40 nm that harbor DNA (CsNIV) or RNA genomes (RsRNAV, HcRNAV) were comparable to or lower than most probable number (MPN) values, which indicate only infectious virus number, suggesting that the FCM counts were underestimates. This is presumably because their single particle fluorescence signals were below the detection limit for the flow cytometer. These results indicate that a large portion of the smaller viruses in the aquatic plankton virus community may be overlooked by FCM.

[1]  K. Tarutani,et al.  Isolation and characterization of two distinct types of HcRNAV, a single-stranded RNA virus infecting the bivalve-killing microalga Heterocapsa circularisquama , 2004 .

[2]  K. Nagasaki,et al.  Genomic and phylogenetic analysis of a single-stranded RNA virus infecting Rhizosolenia setigera (Stramenopiles: Bacillariophyceae) , 2006, Journal of the Marine Biological Association of the United Kingdom.

[3]  G. Bratbak,et al.  Flow cytometric detection of viruses. , 2000, Journal of virological methods.

[4]  G. Bratbak,et al.  High abundance of viruses found in aquatic environments , 1989, Nature.

[5]  C. Brussaard,et al.  Viral Control of Phytoplankton Populations—a Review1 , 2004, The Journal of eukaryotic microbiology.

[6]  K. Nagasaki,et al.  Comparison of Genome Sequences of Single-Stranded RNA Viruses Infecting the Bivalve-Killing Dinoflagellate Heterocapsa circularisquama , 2005, Applied and Environmental Microbiology.

[7]  A. M. Chan,et al.  CHARACTERIZATION OF HaRNAV, A SINGLE‐STRANDED RNA VIRUS CAUSING LYSIS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) 1 , 2003 .

[8]  S. Pietrokovski,et al.  Algal Viruses with Distinct Intraspecies Host Specificities Include Identical Intein Elements , 2005, Applied and Environmental Microbiology.

[9]  G. Bratbak,et al.  Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry , 1999, Applied and Environmental Microbiology.

[10]  C. Brussaard.  Optimization of Procedures for Counting Viruses by Flow Cytometry , 2004, Applied and Environmental Microbiology.

[11]  Feng Chen,et al.  Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold , 2001, Applied and Environmental Microbiology.

[12]  R. Sandaa,et al.  Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla. , 2004, Virology.

[13]  C. Suttle.  Viruses in the sea , 2005, Nature.

[14]  N. Kurano,et al.  Calculation of Most Probable Number for Enumeration of Bacteria on a Micro-Computer , 1986 .

[15]  J. Fuhrman.  Marine viruses and their biogeochemical and ecological effects , 1999, Nature.

[16]  K. Wommack,et al.  Virioplankton: Viruses in Aquatic Ecosystems , 2000, Microbiology and Molecular Biology Reviews.

[17]  K. Nagasaki,et al.  Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera , 2004, Applied and Environmental Microbiology.

[18]  K. Nagasaki,et al.  Previously Unknown Virus Infects Marine Diatom , 2005, Applied and Environmental Microbiology.